Culture vessel

ABSTRACT

Apparatus for growing cells or tissue in vitro and for supporting, positioning and/or centering cell culture inserts that are used in such procedures. The apparatus comprises a body with a plurality of wells wherein a cell culture insert may be supported and centered without interfering with the growing of tissue culture in vitro.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to an apparatus useful for procedures in growingcells or tissue culture in vitro and more particularly for supporting,positioning and/or centering cell culture inserts that are used in theprocedures.

2. Description of the Related Art

Culture vessels are described in U.S. Pat. Nos. 4,495,289 and 5,026,649and European Patent Application No. 0 483 620 A2. Culture vesselscomprise wells which generally have a circular shape and size whichpermits the introduction therein of a cell culture insert having amembrane upon which cell attachment, growth and differentiation occur.The culture vessels containing the wells are typically rectangular andhave a standard size in order to accommodate standard analyticalapparatus.

Conventional cell culture inserts used in culture vessels are describedin U.S. Pat. Nos. 4,871,674 and 5,026,649. U.S. Pat. No. 4,871,674discloses a cell culture insert which comprises discontinuous projectingparts for hanging the cell culture insert on an upper circumferentialpart of the culture vessel. U.S. Pat. No. 5,026,649 discloses a cellculture insert which comprises a projecting part for hanging the culturecell on an upper circumferential part of the culture vessel, and furtherincludes openings in the sidewalls for exchanging gas and for pipetingsample.

Although there are a number of culture vessels and cell culture insertscommercially available and described in patent publications, it isbelieved that there are no culture vessels or cell culture insertsavailable that can be used together to support, position and/or center acell culture insert without interfering with the growing of tissuecultures in vitro.

SUMMARY OF THE INVENTION

The present invention is a device for growing cells or tissue culture invitro. The device comprises a body with wells shaped to accommodate aninsert, such as a cell culture insert.

The device preferably comprises a test plate including an upper surface,a lower surface, and a plurality of wells. Each well is substantiallydisposed between the upper and lower surface of the test plate. Eachwell comprises a sidewall, a bottom surface and a raised mouth surface.Most preferably, the raised mouth surface of each well includes a meansfor supporting a cell culture insert. This means is desirably raisedfrom the upper surface.

The raised mouth surface preferably includes means for positioningand/or centering a cell culture insert. The preferred means is at leastone integral lip portion extending from the raised mouth surface. Theintegral lip portion may be arranged to assist in positioning and/orcentering a cell culture insert without limiting the movement of thecell culture insert.

Desirably, the cell culture insert that may be used with the devicecomprises an upper portion, a lower portion and sidewalls extending fromthe upper portion to the lower portion. The lower portion includes abottom surface which may have attached thereto a flat permeablemembrane. The upper portion includes a top surface which carries atleast one outwardly extending flange. The flange allows the cell cultureinsert to be supported on the top surface of the well of the device sothat there is clearance between the bottom of the membrane and thebottom surface of the well. The clearance provides a controlled statichead and diffusion for the fluid in the device so that cells can beproperly cultured.

The cell culture insert may further include at least one support footingon the bottom surface. The footing may provide a degree of clearancebetween the membrane and the bottom surface of the well.

Most preferably, the outer surface of the sidewall of the cell cultureinsert may further include means for restricting and/or preventing theflange of the cell culture insert from falling into the well of thedevice.

Although the device of the present invention may be configured in acircle with one well, the preferable form of the device is a multi-sidedstructure with wells in an ordered array of parallel rows. Furthermore,the integral lip portions on the raised mouth surface of each well maybe positioned so that the integral lip portion of one well does not facean integral lip portion of an adjacent well. This positioning of theintegral lip portions assures that the flanges of adjacent cell cultureinserts positioned in the wells can be accessible, do not contact oneanother and that the cell culture insert may be aseptically placed andremoved into or out of the well of the device.

The device may further include a removable lid which can be positionedover the upper surface. The lid includes a top wall and a peripheralskirt that extends from the top wall. The lid serves to prevent the lossof sample from the well interior, to protect the contents of the devicefrom the environment and to protect the user from the contents of thedevice should it contain a harmful or potentially harmful material.

The device preferably receives a cell culture, insert in the well. Thecell culture insert is positioned into the well wherein the flanges ofthe cell culture insert are supported on the raised mouth surface of thewell and located between the integral lip portions. The integral lipportions assist in substantially positioning and/or centering the cellculture insert in the well and the raised mouth surface of the wellsubstantially restricts the cell culture insert from falling into thewell. Movement of the cell culture insert within the well may be suchthat the cell culture insert wall and the substantially parallel wellsidewall can touch at one or more locations.

The exterior dimensions of the portion of the cell culture insert withinthe well of the device are sufficiently less than the well diameter toallow a pipet or similar device to be positioned in the space betweenthe well of the device and the cell culture insert for fluid filling oraspiration without disturbing or removing the cell culture insert fromthe well of the device. The space allows the pipet to reach the bottomof the well and introduce or remove medium from beneath the membrane andabout the outer surface of the sidewall of the cell culture insertwithout contaminating the upper surface of the membrane.

The device and related removable lid may be formed in different sizesand geometric configurations so as to be used with different size andgeometric configured cell culture inserts. The removable lid may beformed to be positioned over the upper surface of the device in oneorientation so as to reduce cross contamination between the wells in theevent the lid is repositioned over the upper surface of the device. Thedevice and the removable lid are preferably made of an optically clearplastic to facilitate viewing of the wells and cell culture inserts.

An important feature of the present invention is that in a multi-wellconfiguration, flange to flange contact between the cell culture insertsis prevented so as to reduce contamination. This is accomplished due tothe orientation of the integral lip portions on each well wherein theintegral lip portion of one well does not face or interfere with anintegral lip portion of an adjacent well.

A further feature of the present invention is that the top surfaceconfiguration of the well of the device may facilitate the asepticplacement and removal of the cell culture insert into or out of the wellof the device with tweezers or forceps, as well as providing support tothe cell culture insert when placed in the well of the device.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded perspective view of a multi-well culture vesseland a cell culture insert.

FIG. 2 is a cross sectional view taken along lines 2--2 of FIG. 1illustrating the means available for supporting, positioning and/orcentering a cell culture insert in the well of a culture vessel.

FIG. 3 is an exploded perspective view of a multi-well culture vesseland the optional removal lid not attached.

FIG. 4 is a top view of FIG. 1 illustrating the cell culture insertsupported in the well of a culture vessel.

FIG. 5 is a cross-sectional view taken along lines 5--5 of FIG. 4illustrating where a pipet tip, shown in phantom, may enter the spacebetween the cell culture insert and the well of the culture vessel.

FIG. 6 is an exploded perspective view of an alternate embodiment of thepresent invention, a one-well culture vessel.

DETAILED DESCRIPTION

An apparatus 10 for growing tissue cultures as shown in FIG. 1 includesa culture vessel 15 and a cell culture insert 40. Although a six wellculture vessel is shown, it should be appreciated that the culturevessel may have one, eight, twelve, twenty-four or some other number ofwells selected for the particular purpose for which the apparatus systemis used.

As shown in FIGS. 1 and 2, culture vessel 15 includes a base 11 with anupper surface 12 and lower surface 13. The culture vessel furtherincludes a number of wells 14 each comprising a sidewall 20 extendingfrom the upper surface to the lower surface. A top portion 19 of thewell comprises an open end 22, a top surface 24 extending from the uppersurface and two integral lip portions 26 extending from the top surface.A bottom portion 18 of the well comprises a closed end 21.

Base 11, as illustrated in FIG. 1, typically is transparent and may bemolded, for example, of polyvinylchloride or polystyrene. The culturevessel further includes sidewalls 27, 28, 29, 30, 31 and 32 that extendfrom upper surface 12 to lower surface 13. Sidewalls 27 and 29, and 28and 31 are substantially parallel to each other respectively. Sidewall30 is located between sidewalls 29 and 31 and sidewall 32 is locatedbetween sidewalls 31 and 27.

As shown in FIG. 1, the cell culture insert includes a body wall 42having an outer wall surface 44 and an inner wall surface 46. Body wall42 extends from an upper portion 48 to a lower portion 50. The body wallmay taper from the upper portion to the lower portion. Upper portion 48comprises a top surface 52 which carries two outwardly extending flanges54. The flanges are located opposite from one another approximately 180°apart and are discontinuous. Flanges 54 comprise a horizontalorientation for resting on top surface 24 of well 14.

Lower portion 50 comprises a bottom surface 56 to which a microporousmembrane 58 is adhered. The membrane may be made of suitable materialincluding, but not limited to, perforated inert film, hydrated gel, or alayered combination.

In the embodiment illustrated, two projecting or extending tabs 60 arelocated on outer wall surface 44 of the cell culture insert. The tabsare located opposite from one another or approximately 180° apart andapproximately half the distance between the upper and lower portions.The projecting tabs provide stability to the cell culture insert when itis in the well and shifted for pipette insertion. In the event the cellculture insert is shifted and one of the flanges falls into the well,the projecting tabs will substantially prevent the opposite flange fromfalling into the well.

The cell culture insert may further include feet or supports 61 locatedon the bottom surface. The feet are located opposite from one another orapproximately 180° apart. Feet 61 are used when the extending flanges donot provide adequate support to the cell culture insert in a particularwell configuration. It is most desirable that a space be maintainedbetween the membrane on the bottom surface of the cell culture insectand the well of the culture vessel so that cells may be cultured on bothsides of the membrane. Feet 61 further provide support to the cellculture insert when placed on a flat surface.

As shown in FIG. 3, the apparatus further includes a removable lid 70.Removable lid 70 includes a top wall 72, and peripheral sides 75, 76,77, 78, 79 and 80 that extend from the top wall. The lid removablycovers the upper surface of the culture vessel wherein the peripheralsides abut closely with the base of the culture vessel. In particularperipheral sides 77 and 79 of the lid abut with sidewalls 30 and 32 ofthe culture vessel. The lid is configured so that peripheral sides 77and 79 are unable to abut with sidewalls 27, 28, 29 and 31 of theculture vessel. Therefore the lid may only be matted with the culturevessel in one way so that cross contamination is minimal between thewells of the culture vessel.

As shown in FIG. 4, wells 14 are sufficiently spaced from one anotherand the integral lip portions are oriented so as not to face orinterfere with the integral lip portions of adjacent wells. The integrallip portions of each well are located opposite from one another orapproximately 180° apart. This particular orientation of the integrallip portions substantially prevents flange to flange contact between thecell culture inserts and substantially reduces cross contaminationbetween the cell culture inserts.

FIG. 5 illustrates the orientation of the cell culture insert assupported in culture vessel 15 wherein flange 54 is positioned on topsurface 24 and between integral lip portions 26. Top surface 24 supportsthe cell culture insert and integral lip portions 26 assist inpositioning and/or centering the cell culture insert without limitingmovement of the cell culture insert. FIG. 5 also illustrates a pipet tip64, in phantom, which enters the space 62 between outer wall surface 44of the cell culture insert and sidewall 20 of the culture vessel.

The centering feature provided by integral lip portions 26 can be seenin FIG. 5. Cell culture insert 40 is set within well 14 and spacedsufficiently from well sidewall 20 as facilitated by top surface 24 andintegral lip portions 26. Thus, capillary action should not occur tocause solution or media in space 62 from wicking up outer surface 44 andentering the interior of the cell culture insert or spilling from well14. The movement of the cell culture insert, however, may be such thatthe cell culture outer wall surface and the sidewall of the well cantouch at one or more locations.

FIG. 6 illustrates a further embodiment of the present invention whereinthe culture vessel includes one well. FIG. 6 includes many componentswhich are substantially identical to the components of FIGS. 1-4.Accordingly, similar components performing similar functions will benumbered identically to those components of FIGS. 1-5, except that asuffix "a" will be used to identify those similar components in FIG. 6.

As illustrated in FIG. 6, a further embodiment of the invention includesa one well apparatus 90 having a culture vessel 100, a cell cultureinsert 40a and a removable lid 70a. The culture vessel includes a base102 with an upper surface 104 and a lower surface 105. A well 106comprising a sidewall 107 extends from the upper surface to the lowersurface. A top portion 108 of the well comprises an open end 110, a topsurface 112 extending from the upper surface and two integral lipportions 114 extending from the top surface. A lower portion 106 of thewell comprises a closed end 118.

Base 102 typically is transparent and may be molded, for example, ofpolyethylene terephthalate. The culture vessel further includessidewalls 120, 121, 122, 123, 124 and 125 that extend from upper surface104 to lower surface 105.

As practitioners-in-the-art will understand, the culture vessel and cellculture insert of the present invention may be comprised of simplemoldable parts which may be mass produced from a variety of materials,including, for example, polyethylene, polystyrene, polyethyleneterephthalate, and polypropylene. As will be understood further bypractitioners in the art, materials should be selected which provide asmall degree of resiliency for the purpose of providing ease ofinsertion of the cell culture inserts into the culture vessel and easeof use for subsequent examination of the developed cultured cells.

What is claimed is:
 1. Apparatus for use in growing tissue cultures invitro comprising:a culture vessel comprising an upper surface, a lowersurface, a plurality of wells extending from said upper surface to saidlower surface, a raised mouth surface extending from said upper surfaceof each well for supporting a cell culture insert and projectingportions extending from said raised mouth surface of each well forpositioning a cell culture insert; and a cell culture insert suspendedin one of said wells and comprising an upper portion, a lower portion, asidewall comprising an inner and outer surface and extending from saidupper portion to said lower portion, a permeable membrane attached tosaid lower portion means for suspending said insert in said one of saidwells, and means for restricting, movement of said cell culture insertin said one of said wells of said culture vessel extending from saidouter surface of said sidewall wherein said means for suspending ispositioned on said raised mouth portion between said projecting portionsto position said insert.
 2. The apparatus of claim 1 wherein saidprojecting portions comprise two projecting portions extending from saidraised mouth surface of each well and being located opposite from oneanother.
 3. The apparatus of claim 1 wherein said culture vessel issubstantially rectangular in shape and said wells are in an orderedarray of substantially parallel rows.
 4. The apparatus of claim 1wherein said means for suspending said insert includes at least onediscontinuous projecting flange extending from said upper portion. 5.The apparatus of claim 4 wherein said means for restricting movement ofsaid insert includes at least one projecting tab located on the outersurface of said sidewall and being constructed so as to provide accessof sufficient size between said outer sidewall of said insert and thewall of said one of said wells of said culture vessel so as tosubstantially prohibit capillary activity.
 6. The apparatus of claim 1wherein said means for suspending said insert includes two discontinuousprojecting flanges located opposite from one another and extending fromsaid upper portion.
 7. The apparatus of claim 6 wherein said means forrestricting movement of said insert includes two projecting tabs locatedopposite one another on the outer surface of said sidewall and betweensaid upper portion and said lower portion.
 8. The apparatus of claim 6wherein said projecting portions of the wells are in an ordered array tosubstantially prevent the flange of one cell culture insert fromcontacting the flange of another cell culture insert.